Participants A total of 104 consecutive patients with acute
coronary syndromes (42 with unstable angina and 62 with AMI), and 64
patients with stable coronary artery disease (CAD) without evidence of
ischemia.

Main Outcome Measures Ability of MDA-modified LDL, C-reactive
protein, and troponin I to discriminate patients with stable CAD,
unstable angina, or AMI.

Results Malondialdehyde-modified LDL
(χ2=10.2;
P=.001), but not troponin I or C-reactive
protein, discriminated between stable CAD and unstable angina. Troponin
I (χ2=14.5; P<.001), but not
MDA-modified LDL or C-reactive protein, discriminated between unstable
angina and AMI. Both MDA-modified LDL and troponin I
(χ2=14.5; P<.001 and
χ2=5.3; P=.02,
respectively) but not C-reactive protein discriminated between stable
CAD and AMI. The sensitivity of MDA-modified LDL was 95% for unstable
angina and 95% for AMI, with a specificity of 95%. Values for
troponin I were 38% and 90%, respectively, with a specificity of
95%. The combination of MDA-modified LDL and troponin I had a
sensitivity of 98% for unstable angina and 100% for AMI, with a
specificity of 99%.

Conclusion The combination of MDA-modified LDL, which
may reflect endothelial injury or plaque instability, and troponin I,
which reflects myocardial cell injury, allows better discrimination
between stable CAD and acute coronary syndromes than troponin I
alone.

Ischemic
injury of endothelium is associated with prostaglandin synthesis and
platelet adhesion and aggregation, which may be associated with the
release of aldehydes that substitute the lysine residues in the
apolipoprotein B-100 moiety of low-density lipoprotein (LDL) in the
absence of lipid peroxidation.1,2 This type of oxidatively
modified LDL is referred to as malondialdehyde (MDA)-modified
LDL.3 Plasma levels of MDA-modified LDL are increased in
patients with acute coronary syndromes, ie, unstable angina or acute
myocardial infarction (AMI).3,4 Previously, C-reactive
protein5 and cardiac troponins6- 9 have been
proposed as diagnostic markers of acute coronary syndromes. In this
study, we compared the usefulness of MDA-modified LDL as a marker of
acute coronary syndromes with that of C-reactive protein and troponin
I.

METHODS

A total of 168 patients were studied, including 104 consecutive
patients admitted to the emergency department of the University
Hospital of Leuven, Belgium, because of an acute coronary syndrome and
64 patients with stable, angiographically documented coronary artery
disease (CAD) without clinical signs of ischemia. Patients with stable
CAD were treated with aspirin, β-blockers, nitrates, and, in a great
majority, angiotensin-converting enzyme inhibitors. Patients with acute
coronary syndromes had ischemic chest discomfort with transient
ST-segment elevation or persistent ST-segment depression of more than
0.5 mm, or T-wave inversion of more than 1 mm. Blood samples for
MDA-modified LDL assay from patients with acute coronary syndromes were
collected at admission, within 6-8 hours of onset of chest pain, and
before therapy was started. Blood samples for total creatine kinase
(CK) and for CK isoenzyme MB mass assay were obtained at entry, 6 to 8
and then 12 to 16 hours after admission. Enzyme evidence of AMI was
defined as CK-MB elevation above the normal range and at least 3% of
total CK. All patients with acute coronary syndromes received oral
aspirin, intravenous nitrates, and heparin for 48 to 72 hours. Most of
these patients underwent coronary angiography and revascularization
within the first 3 to 5 days after hospital admission. Blood samples
were collected in
the fasting state in patients with stable CAD.

Venous blood samples were collected in 0.1 volume of 0.1 mol/L of
citrate, containing 1 mmol/L of EDTA, 20 µmol/L of vitamin E, 10
µmol/L of butylated hydroxytoluene, 20 µmol/L of dipyridamole, and
15 mmol/L of theophylline. Blood samples were centrifuged at
3000g for 15 minutes at room temperature within 1 hour after
collection and stored at −30°C until assays were
performed.3,4,10

A monoclonal antibody 1H11–based competition enzyme-linked
immunosorbent assay (ELISA) was used for the quantitation of
MDA-modified LDL in plasma.3,4 The C50 values, ie, concentrations that are required to obtain 50% inhibition of
antibody binding to immobilized in vitro MDA-modified LDL in the ELISA,
are 0.65 mmol/L (25 mg/dL) for native LDL, 0.001 mmol/L (0.05 mg/dL)
for MDA-modified LDL with at least 60 aldehyde-substituted lysines per
apolipoprotein B-100, and 0.06 mmol/L (2.5 mg/dL) for oxidized LDL with
fragmented apolipoprotein B-100 moiety that was generated via copper
ion–induced lipid peroxidation. Intra-assay and interassay
coefficients of variation were 12% and 15%, respectively. When in
vitro MDA-modified LDL was added to control human plasma at a final
concentration of 0.006 mmol/L (0.25 mg/dL), recovery was 95%. Ten
plasma samples from AMI patients were thawed, assayed, and frozen
again. The MDA-modified LDL levels (SDs) in these samples were 0.04
(0.003) mmol/L (1.4 [0.1] mg/dL) after the first thawing, 0.03
(0.005) mmol/L (1.3 [0.2] mg/dL) after the second thawing, and 0.04
(0.005) mmol/L (1.5 [0.2] mg/dL) after the third thawing. Levels of
MDA-modified LDL were measured blindly at the University of Leuven's
Center for Molecular and Vascular Biology, where the test was
developed.

Total and high-density lipoprotein (HDL) cholesterol and
triglyceride levels were measured by enzymatic methods (Boehringer
Mannheim, Mannheim, Germany). Low-density lipoprotein cholesterol
levels were calculated with the Friedewald formula. Creatine kinase
activity and CK-MB mass were measured using commercially available
assays (Boehringer Mannheim and Abbott Laboratories, North Chicago,
Ill, respectively). C-reactive protein levels were measured with a
commercial immunoassay (Boehringer Mannheim). All measurements
were performed at the clinical laboratory of the University Hospital in
Leuven. Cardiac troponin I levels were measured on a Beckman ACCESS
immunoanalyzer (Analis, Gent, Belgium) using commercially available
monoclonal antibodies (Sanofi, Toulouse, France). These measurements
were performed in the laboratories of Analis.

Plasma levels of total cholesterol, HDL cholesterol, LDL
cholesterol, triglycerides, MDA-modified LDL, C-reactive protein, and
troponin I were compared in patients with stable CAD and those with
acute coronary syndromes by nonparametric Kruskal-Wallis test followed
by the Dunnett multiple-comparison test. Discontinuous parameters were
compared by χ2 analysis. Logistic regression models were
used to determine the associations between acute coronary syndromes and
C-reactive protein, cardiac troponin I, and MDA-modified LDL. For
continuous variables, cubic spline functions were used to model the
relationship between covariates and response. Multiple receiver
operating characteristic (ROC) curve analysis11 was
performed to determine if C-reactive protein, troponin I, or
MDA-modified LDL could discriminate among stable CAD, unstable angina,
and AMI. All analyses were performed with SPlus, Version 4.0 (Mathsoft
Inc, Cambridge, Mass) and SAS/STAT, Version 6.12 (SAS Institute Inc,
Cary, NC). P<.05 was considered statistically significant.

RESULTS

Of the 104 patients with acute coronary syndromes, 62 had elevated
CK-MB levels indicative of AMI. In 42 patients, no CK-MB elevations
were found, and these patients were classified as having unstable
angina pectoris. Patients with stable CAD and those with acute coronary
syndromes were
similar with respect to age and sex as well as
serum levels of total cholesterol, LDL cholesterol, HDL cholesterol,
and triglycerides (Table 1).

Compared with patients with stable CAD, C-reactive protein levels were
2.4-fold higher in patients with unstable angina
(P=.02) and 3.5-fold higher in patients with
AMI (P<.001). Plasma levels of C-reactive protein were
somewhat higher (P=.02) in patients with AMI
than those with unstable angina (Table 1). Compared with patients with
stable CAD, troponin I levels were 5.4-fold higher in patients with
unstable angina (P<.001) and 26-fold higher in patients with
AMI (P<.001). Plasma troponin I levels were significantly
higher in patients with AMI than those with unstable angina
(P<.001) (Table 1). Compared with patients with stable CAD,
plasma levels of MDA-modified LDL were 2.9-fold higher in patients with
unstable angina (P<.001) and 2.6-fold higher in patients
with AMI (P<.001). Plasma levels of MDA-modified LDL were
similar in patients with unstable angina and those with AMI
(P=.09).

Multiple ROC analysis revealed that MDA-modified LDL
(χ2=10.2;
P=.001), but not troponin I and C-reactive
protein, discriminated between stable CAD and unstable angina. In
contrast, troponin I (χ2=14.5;
P<.001), but neither MDA-modified LDL nor C-reactive
protein, discriminated between unstable angina and AMI. Both
MDA-modified LDL (χ2=14.5;
P<.001) and troponin I
(χ2=5.3; P=.02),
but not C-reactive protein, discriminated between stable CAD and AMI.

As shown in Table 2, at a cutoff
value of 10 mg/dL (the value exceeding the 95th percentile of
distribution in patients with stable angina), the sensitivity of
C-reactive protein was 19% for unstable angina and 42% for AMI,
whereas the specificity was 95%. At a cutoff value of 0.05 µg/L (the
value exceeding the 95th percentile of distribution in patients with
stable angina), the sensitivity of troponin I was 38% for unstable
angina and 90% for AMI, whereas the specificity was 95%. At a cutoff
value of 0.02 mmol/L (0.70 mg/dL; the value exceeding the 95th
percentile of distribution in patients with stable angina), the
sensitivity of MDA-modified LDL was 95% for unstable angina and 95%
for AMI, and the specificity was 95%.

The study cohort included 98 patients (35 with stable CAD, 18 with
unstable angina, and 45 with AMI) who have been described
elsewhere4 and 70 new patients (29 with stable CAD, 24 with
unstable angina, and 17 with AMI). The age; sex; serum levels of total
cholesterol, LDL cholesterol, HDL cholesterol, and triglycerides; and
levels of C-reactive protein, troponin I, and MDA-modified LDL of
patients with stable angina and acute coronary syndromes were similar
in the previously reported subjects and the overall study cohort, as
were the sensitivities for troponin I and MDA-modified LDL for unstable
angina and AMI.

As shown in Table 3, when
MDA-modified LDL and troponin I were combined, the sensitivity was 98%
for unstable angina compared with 95% when MDA-modified LDL was used
alone and 38% when troponin I was used alone. When MDA-modified LDL
and troponin I were combined, the sensitivity was 100% for AMI
compared with 95% for MDA-modified LDL alone and 90% for troponin I
alone. The specificity increased from 95% for MDA-modified LDL or
troponin I alone to 99% for their combination.

COMMENT

We compared the usefulness of MDA-modified LDL for the diagnosis of
unstable angina and AMI with C-reactive protein and cardiac troponins,
which have been proposed as diagnostic markers for acute coronary
syndromes.5- 9 Mach et al12 reported increased
levels of C-reactive protein in only 5% of patients with unstable
angina. Hamm et al13 found positive troponin I test results
in 36% of patients with unstable angina, whereas Galvani et
al14 found positive troponin I test results in only 24% of patients with unstable angina. In the present study, the rather low
diagnostic values of C-reactive protein and troponin I for unstable
angina were confirmed: their sensitivities were 19% and 38%,
respectively. However, troponin I was found to be a clinically useful
marker for AMI—the sensitivity was 90% compared with 100% reported
by Hamm et al.13

Because an increase of troponin I depends on myocardial cell
injury, it is logical to assume that troponin I is a better marker for
AMI than for unstable angina. We hypothesized that a parameter that has
been associated with ischemic injury of endothelium, plaque
instability, or both, which precede cardiac necrosis, would be a more
sensitive marker for unstable angina than troponin I. Multiple ROC
analyses showed that MDA-modified LDL better discriminated between
stable CAD and unstable angina than troponin I. The sensitivity of
MDA-modified LDL for unstable angina was 95% compared with 38% for
troponin I. The similar sensitivities of MDA-modified LDL for unstable
angina and AMI suggest that it may be a clinically useful marker of
acute coronary syndromes irrespective of the occurrence of myocardial
cell injury.

Endothelial injury in association with hypoxia may result in the
activation of not only the cyclooxygenase-dependent pathway of
prostaglandin synthesis in endothelial cells15 but also in increased production of F2α-isoprostanes,
noncyclooxygenase-derived prostaglandin-like compounds1,16
that are strong inducers of platelet activation. Plaque instability is
associated with increased platelet adhesion and activation. Activated
platelets may then produce large amounts of aldehydes, further
enhancing the modification of LDL. The association of unstable angina
and AMI with plasma levels of MDA-modified LDL supports the hypothesis
that the generation of MDA-modified LDL is associated with ischemic
injury or plaque instability rather than with the extent of coronary
atherosclerosis. The low reactivity of the monoclonal antibody 1H11
with nonthrombotic atherosclerotic plaques, in contrast with the high
reactivity with unstable plaques, suggests that MDA-modified LDL, in
contrast with oxidized LDL, is not released continuously from
atherosclerotic plaques but is generated in unstable
plaque.4

In conclusion, this study shows that the combination of MDA-modified
LDL, which may reflect endothelial injury or plaque
instability,3,4 and troponin I, which reflects myocardial
cell injury,13 allows a more sensitive and specific
discrimination between stable CAD and acute coronary syndromes than
troponin I alone. Further evaluation of the prognostic value of
MDA-modified LDL for AMI in patients with unstable angina requires
large-scale prospective studies.

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